EXPERIMENTAL AND THERAPEUTIC 21: 456, 2021

Congenital dyserythropoietic and drug‑induced liver injury present as bland cholestasis: A case report

YUE HAN1,2*, YAN ZHUANG1*, WEILIANG TANG1*, LU CHEN1*, YEJING CHEN3, QIMING GONG1 and XINXIN ZHANG1,2,4

1Department of Infectious Diseases, Research Laboratory of Clinical Virology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine; 2Sino‑French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai 200025; 3Department of Infectious Diseases, Qionghai People's Hospital, Hainan 571400; 4Clinical Research Center, Ruijin Hospital North Campus, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China

Received November 2, 2020; Accepted February 2, 2021

DOI: 10.3892/etm.2021.9887

Abstract. and drug‑induced liver injury(DILI) Introduction are separate disorders, which are difficult to diagnose. The clinical effects of DILI vary among individuals. However, Hereditary anemias are a group of common but highly pleio‑ the outcome determinants remain to be fully established. To tropic disorders, exhibiting great molecular disparity. As the best of our knowledge, the role of anemia in DILI has yet highly heterogenous hematological disorders (1), the clinical to be reported. The present study reported on the case of one knowledge of hereditary anemias remains poor. To date, Chinese patient (male; age, 21 years) who experienced obvious ≥70 genes have been identified to be involved in anemia (2). drug‑induced cholestasis. Of note, the hepatocyte injury was in these genes occur with low to rare frequency. minimal compared with that in previously reported cases The mechanisms underlying the onset of anemias include treated with the same drug. In addition, the patient suffered deficiencies in regeneration capacity, red cell (RBC) from mild with no obvious cause. A genetic deformities, enzymatic deficiencies and other aspects of pedigree analysis revealed compound heterozygous mutations hematopoiesis. Precise diagnosis is of paramount importance, in the congenital anemia‑associated gene codanin 1, including since the therapeutic approaches for anemias and contrain‑ the novel rare p.R1067H . Treatment with urso‑ dications are distinctly different (3). Furthermore, verifying deoxycholic acid alone sufficed and the outcome was good. genetic defects in adults is always challenging, largely due to Therefore, whilst chronic predisposed the liver the low penetrance rate, pleiotropic expression and multifacto‑ to cholestasis, it could have shielded the liver from further rial mechanisms. Therefore, the diagnosis and management of injuries, since , a by‑product of hemolysis, is a known anemia remain challenging. antioxidant. The results of the present study indicated that Drug‑induced liver injury (DILI) is a potentially serious genetic screening may be used for the diagnosis of liver injury adverse reaction (4,5). Current diagnostic scales heavily concurring with undiagnosed anemia. rely on causality assessment, such as the Roussel Uclaf Causal Relationship Assessment Method (RUCAM) scoring system (6), partially due to the lack of knowledge on its determinants. Downstream intrahepatic cellular events such as may aggravate liver injury following the initial insult (7,8). The of DILI may mimic almost any other liver disorder. The clinical presentation may vary significantly among treated patients, even following treatment Correspondence to: Professor Qiming Gong or Professor with the same substance. Therefore, determining protective Xinxin Zhang, Department of Infectious Diseases, Research and predisposing factors remains difficult. The overlap of Laboratory of Clinical Virology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Building 36, 197 Ruijin DILI with a rare form of hereditary anemia has not been previ‑ Er Road, Shanghai 200025, P.R. China ously reported. The present study reported on a case of DILI E‑mail: [email protected] presenting with bland cholestasis in the context of a chronic E‑mail: [email protected] rare anemia. The potential interactions between these two disorders were evaluated and discussed. *Contributed equally Materials and methods Key words: rare congenital anemia, cholestasis, hyperbilirubinemia, ephedra‑induced, protective Clinical presentation. An otherwise healthy 21‑year‑old male was initially admitted to Ruijin Hospital (Shanghai, China) in March 2020 due to afebrile painless for 20 days. The 2 HAN et al: CONGENITAL ANEMIA AND CHOLESTASIS patient was in a normal state until he underwent laparoscopic (H&E) and Masson trichrome (11) using standard protocols. cholecystectomy after several episodes of abdominal and Liver biopsy sample was fixed in 10% neutral phosphate cholelithiasis in September 2019. No transfusion was required buffered formalin at room temperature for 2 h. Xylene and during the . Ιn October 2019, the patient developed ethanol gradient method was applied in the and generalized pruritus of unclear etiology. The patient under‑ rehydration process. Paraffin was used as embedding reagent went self‑treatment with ephedra (9), which slightly improved and the thickness of slices was 3‑5 µm. The cells were stained the symptoms. In the meantime, the patient also complained sequentially with hematoxylin and eosin at room temperature of fatigue and anorexia. The patient noticed yellowish eyes for 4 and 10 min, respectively. The slides were observed under and dark urine, which prompted a clinical evaluation. In addi‑ x100 and x400 magnification using light microscopy. Slides tion, the patient reported pale stool, which was not observed were evaluated by one experienced pathologist who was during hospitalization. The paternal grandfather of the patient blinded to the clinicopathological data of the patient. had died of cirrhosis at 36 years of age due to unknown etiology. Apart from the above medical and surgical history, Treatment and follow‑ups. The patient was treated with urso‑ the patient's medical history was unremarkable. The patient deoxycholic acid (UDCA) 250 mg t.i.d. po, starting on day 4 had no and did not smoke or drink alcohol. On from hospitalization, for one month. At 3 and 6 months after arrival, the patient's vital signs were stable and normal and discharge, follow‑up checks, including abdominal , he was conscious. On examination, the patient had icterus , liver and renal tests, were performed. with jaundiced skin, with no visible lesions. The abdomen was distended and the and liver were non‑palpable below Results the costal margin. The remainder of the physical examination was unremarkable. Written informed consent for data usage in Clinical and laboratory findings. Examination of the other‑ scientific research, including and publication of wise healthy young male patient with jaundice for 20 days the results was provided by the patient and his parents. and pruritus for 6 months following cholecystectomy revealed potential ephedra‑induced liver injury with . In Next‑generation sequencing and Sanger confirmation. Given contrast to the obvious cholestasis, the liver injury was limited the family history of cirrhosis, whole‑exome sequencing of the compared with other reported cases (9,12), where patients were pedigree, including the proband and his parents was performed also self‑treated with the same medication. CT scans indicated to detect any possible genetic abnormalities. Genomic DNA no focal lesions in the liver. Magnetic resonance cholangiopan‑ was extracted using QIAamp DNA Blood Mini Kit (Qiagen, creatography revealed a slightly dilatated post‑cholecystectomy Inc.) from peripheral blood and was randomly fragmented using common bile duct with no evidence of choledocholithiasis Covaris S220 Focused‑ultrasonicator system (Covaris Inc.) (Fig. 1), moderate and intrahepatic to an average size of 180‑280 bp. Libraries were constructed lymphatic stasis. Furthermore, laboratory testing revealed using paired‑end protocols from Illumina, Inc. The fragmented hyperbilirubinemia (total/direct bilirubin, 253.8/134.9 µmol/l; DNA was captured using the Agilent SureSelect All normal range, 4.7‑24/0‑6.8 µmol/l), increased bile acid levels ExonV6 kit (Agilent Technologies Inc.) and sequencing was (338.6 µmol/l; normal range, 1.0‑10.0 µmol/l), and normal performed on the Illumina Novaseq 6000 platform (Illumina prealbumin, alanine transaminase, aspartate aminotransferase, Inc.). Paired‑end 150 bp‑long reads with a coverage of ≥10X gamma‑glutamyl transferase and alkaline phosphatase levels, for 99% of the genome (mean coverage, x100) were gener‑ and international normalized ratio value. ated. Candidate variants, amplified by PCR, were confirmed The model for end‑stage liver disease (MELD) score (13) was by Sanger sequencing according to Applied Biosystems® 17 with an R‑factor value of 0.7. Viral and autoimmune hepatitis 3730/3730xl DNA Analyzers User Guide on the ABI 3730 were excluded. RUCAM scoring did not exclude DILI (6). Of Prism DNA sequencer (Applied Biosystems; Thermo Fisher note, the patient also suffered from chronic and mild hemolytic Scientific, Inc.) using two sets of self‑designed primer pair anemia with no identifiable cause ( level, 109 g/l; ( fi r s t p a i r : 5 ' ‑ G G T ​GAT ​ATC ​TCC ​CCC ​TGC ​TC‑3' and 5'‑GCT ​ normal range, 131‑172 g/l) with an elevated mean corpuscular TACTGC​ TGC​ GAG​ AAC​ TG‑3';​ second pair: 5'‑TTATCC​ TGC​ ​ volume (91 fl), low level (<0.1 g/l; normal range, CCA​AGT​CCC​AG‑3' and 5'‑ATT​TGG​GGG​ATC​AGG​CAA​ 0.3‑2.0 g/l) and moderate elevated percentages of reticulo‑ GA‑3') encompassing the region of interest. cytes (2.5%; normal range, 0.5‑1.5%). Other findings included hyperferritinemia (623 ng/ml), increased transferrin saturation Filtering of rare variants and pedigree analysis. Rare exonic (58.6%) and normal ceruloplasmin levels. RBC size variations variants with a minor frequency of <0.01 were selected may be readily observed under a microscope. During hospi‑ from the 1000 Genomes Project (10), the Exome Sequencing talization, the RBC distribution width (15.3‑17.8%; normal Project (ESP; esp6500siv2, https://esp.gs.washington. range, 11.6‑14.0%) and the percentage of basophils (1.6‑2%; edu/drupal/) and The Genome Aggregation Database (gnomAD normal range; <1.0%) were consistently elevated. The levels release v3.1, https://gnomad.broadinstitute.org/). Known of lactate dehydrogenase (LDH) were marginally increased, pathogenic dominant or recessive variations, homozygous or while those of ammonia were normal. These results indicated compound heterozygous, as well as de novo variants, were all chronic mild to moderate . However, the considered as candidate variations in the pedigree analysis. Coombs tests (IgG and C3d), Isopropanol test, Ham's test, Glucose‑6‑phosphate dehydrogenase (G6PD) deficiency test Liver biopsy. Ultrasound‑guided liver biopsy was performed and pyruvate kinase enzymatic activity were negative for auto‑ and the specimen was stained with hematoxylin and eosin immune hemolytic anemia. Finally, high‑performance liquid EXPERIMENTAL AND THERAPEUTIC MEDICINE 21: 456, 2021 3

Figure 1. CT/MRCP and liver biopsy indicated characteristics of cholestasis with mild inflammatory events. CT scan revealed a slightly enlarged (A) spleen and (B) liver. (C) MRCP and (D) 3D reconstruction indicated slightly dilated intrahepatic bile ducts with no evidence of lithiasis (sequence parameters: T2‑weighted; echo time, 120 msec; repetition time, 1,100 msec). (E) H&E staining of the liver biopsy specimen revealing central lobular focal with mild portal inflammation (magnification, x100). (F) H&E staining indicating cholestasis with dilated canaliculi, bile plug, apoptotic bodies and mild microvesicular steatosis (magnification, x400). (G) The C to T substitution in the CDAN1 gene, causing R1067H, was verified by Sanger sequencing. (H) Sanger sequencing of the wild‑type CDAN1 gene from the mother. CT, computed tomography; MRCP, magnetic resonance cholangiopancreatography; H&E, hematoxylin and eosin; CDAN1, codanin 1.

Figure 2. Dynamic changes in bilirubin and bile acid levels at various time‑points during hospitalization. The arrow indicates the date of anti‑cholestasis treatment. UDCA, ursodeoxycholic acid.

chromatography indicated normal levels of hemoglobin α Disease‑causing variation screening results. In addition to (HbA), HbA2 and , whilst no visible inclusion common homozygous polymorphisms in cholestasis‑asso‑ body was observed inside RBCs. ciated genes, including ATP binding cassette subfamily B member 11 (ABCB11) and tight junction protein 2 (TJP2), Liver biopsy and . Staining of liver specimens with the pedigree genetic study revealed compound heterozygous H&E revealed that the lobular structure remained intact, while variations in the congenital anemia‑causing gene codanin 1 intrahepatocyte cholestasis and bile plug were observed in (CDAN1), including a novel rare mutation of paternal origin, the dilated canaliculus. In addition, a small number of foci namely p.R1067H (ClinVar accession no. SCV001448675). of intralobular necrosis, apoptotic bodies, microvesicular Apart from anemia, defects in the CDAN1 gene have also been steatosis and minimal portal inflammation were observed. associated with jaundice, iron overload, (14) and The Masson trichrome staining was normal. The liver tissues (Table I) (15). All of the aforementioned abnormali‑ were negative for hepatitis B surface and core antigens and ties were present in the patient of the present study. for copper. Staining for cytokeratin 19 revealed slight ductular proliferation. The pathological data suggested that the patient Treatment and outcome. The patient was diagnosed with suffered from acute cholestatic hepatitis, most likely caused drug‑induced bland cholestasis, with iron overload in the by DILI (Fig. 1). context of a rare form of congenital anemia. Since anemia 4 HAN et al: CONGENITAL ANEMIA AND CHOLESTASIS

Table I. Homozygous gene polymorphisms associated with cholestasis and congenital anemia causing mutations.

A, Common cholestasis‑associated polymorphisms

Minor allele frequency ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ Inheritance Gene AA Change GnomAD 1000g_Chinese Disease (ID: OMIM or HGMD) pattern Note

ABCB11 p.V444A 0.568838 0.664452 Cholestasis, benign recurrent Autosomal Co‑existed with another intrahepatic, 2 (605479); recessive heterozygous cholestasis, drug‑induced, cholestasis‑related association with (CM071525) polymorphism, p.A1028A TJP2 p.T909T 0.265188 0.460133 Cholestasis, progressive familial Autosomal ‑ p.A913A 0.264951 0.458472 intrahepatic 4 (615878); recessive hypercholanemia, familial (607748)

B, Congenital anemia‑associated mutations

Minor allele frequency ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ Inheritance Gene AA Change GnomAD 1000g_Chinese Disease (ID: OMIM or HGMD) pattern Note

CDAN1 p.R1067H / Novel Dyserythropoietic anemia, Autosomal Paternal origin congenital, type Ia (224120) recessive co‑existed with another two maternal heterozygous variations with unknown significancea aTwo maternal variations with unknown significance included rs56046122 (intronic) and rs28661826 (exonic, also at exon 24). OMIM, Online in Man; HGMD, Human Gene Mutation Database; AA, amino acid.

was mild, the patient was treated only with UDCA, and an intrahepatic protective molecule shielding the liver from cholestasis and pruritus swiftly subsided 2 weeks later further injury. (Fig. 2). Three and six months after discharge, follow‑up Of note, the clinical examination suggested a rare and mild examinations revealed that the patient remained in a good type of chronic hemolytic anemia. These rare disorders may overall condition. be caused by any genetic defect affecting RBCs, including Diamond‑Blackfan anemia, congenital dyserythropoietic Discussion anemias, , sickle cell disease, enzyme deficiency and red cell membrane disorders. These disorders are charac‑ The present study reported on the case of an otherwise healthy terized by a variable degree of anemia, chronic extravascular young male patient with an episode of cholestasis. According hemolysis, reduced erythrocyte lifespan, splenomegaly, jaun‑ to the LiverTox.nih.gov website (16) (https://www.ncbi.nlm. dice, biliary lithiasis and iron overload (17). Each type may nih.gov/books/NBK548711/), ephedra is known to promote require contradictory therapeutic approaches (3); therefore, the liver injury. However, all reported cases, including hepato‑ precise genetic diagnosis is of great importance. In the current cellular and biliary injury (9), with or without co‑treatment case, a rare genetic variant was identified in the CDAN1 gene. with steroids (12), were more severe compared to the present This mutation is involved in the onset of rare congenital case. A comparison of the present and previously reported dyserythropoietic anemia type I (CDA‑I), which is caused by cases is provided in Table II. The previously reported cases bi‑allelic mutations in the CDAN1 gene, usually accompanied were young adults, which is consistent with the current case. by iron overload. In CDA‑I, liver tests are commonly normal Furthermore, not only was the liver injury much milder in unless severe iron overload is present. However, unconjugated the present case, but a large proportion of the young male bilirubin and LDH levels are usually elevated due to hemo‑ patients' liver functions were also well preserved, despite the lysis and ineffective . It has been reported that fact that accumulated bile salts should have aggravated the abdominal ultrasound in patients with CDA‑I reveals universal hepatobiliary injury due to their inherent detergent activi‑ splenomegaly (14), while 50‑60% of adult patients have gall‑ ties (16). All of the above evidence indicated the presence of stones (15). In the current case, all the aforementioned clinical EXPERIMENTAL AND THERAPEUTIC MEDICINE 21: 456, 2021 5

Table II. Comparison among ephedra‑administered cases.

Item Case by Nadir et al (9) Case by Singh et al (12) Present case

Age (years) 33 24 21 Sex Female Male Male Medication Ephedra Ephedra with steroids Ephedra Pattern Hepatocellular Bland cholestatic Bland cholestatic ALT (U/l) 1586 237 37 ALP (U/l) 175 129 104 Bilirubin (µmol/l) 136.80 906.3 253.8 Prothrombin time (sec) 13.2 NA 12 Jaundice Yes Yes Yes Recovery time (months) 4 3 1

Normal range for ALT (10‑64 IU/l), ALP (38‑126 IU/l), Bilirubin (4.7‑24 µmol/l), Prothrombin time (10‑14 sec). ALT, alanine aminotrans‑ ferase; ALP, alkaline phosphatase; NA, not available.

features were present and the patient had splenomegaly and importance of genetic pedigree analysis, particularly when a a history of gallstones. Furthermore, the dysregulation of given condition is manifested in additional family members. erythropoiesis was evidenced by the marginally elevated In addition, a novel rare mutation in the CDAN1 gene was percentage of relative to the degree of anemia, identified in the present case, enhancing the spectrum of and constantly by the increased number of basophils in the mutations known to be associated with such . peripheral blood of the patient. In this case, genetic testing is recommended, since it may significantly reduce the time Acknowledgements required for accurate molecular diagnosis and prevent inva‑ sive aspiration. To the best of our knowledge, Not applicable. the novel p.R1067H mutation identified in the present study, located at the disease‑related hotspot exon 24 (18), has not been Funding previously reported and is adjacent to the well‑established disease‑related p.D1043V mutation. Computational prediction The present study was supported by grants from the National tools, including SIFT (sift.jcvi.org), PolyPhen‑2 (genetics. Natural Science Foundation of China (grant no. 82002126), the bwh.harvard.edu/pp2), mutation taster (www.mutationtaster. Shanghai Jiao Tong University (SJTU) medical X engineering org), PROVEAN (provean.jcvi.org/index.php), SNPs & GO project (grant no. YG2017MS63) and the Excellent Youth B (snps‑and‑go.biocomp.unibo.it), PANTHER (pantherdb.org), Project (grant no. GCQN‑2017‑B20). PhD‑SNP (snps.biofold.org/phd‑snp/phd‑snp.html), I‑Mutant (folding.biofold.org/cgi‑bin/i‑mutant2.0.cgi) and SNAP (www. Availability of data and materials rostlab.org/services/SNAP), unanimously support the delete‑ rious effect of mutations of this gene (19‑21). The next‑generation sequencing datasets generated and While the hemolytic process may predispose the patient's analyzed during the current study are available in the National liver to cholestatic injury, most likely through iron overload, Center for Bioinformatics Information (NCBI) Sequence Read lithiasis and splenomegaly, it may also guarantee a steady flow Archive repository (accession no. PRJNA690696; https://www. of slightly elevated bilirubin in the blood (hyperbilirubinemia). ncbi.nlm.nih.gov/sra/PRJNA690696). Bilirubin, a pigment with potent anti‑oxidative proper‑ ties (22), is known to protect against inflammation‑mediated Authors' contributions diseases (23,24) all‑cause mortality (25) and even possesses therapeutic value (23,24,26). However, the intrahepatic YH drafted the manuscript and performed the laboratory protective role of mildly‑elevated unconjugated bilirubin tests. YZ, WT, LC and YC performed the follow‑up checks has not been reported in the literature, to the best of our and analyzed the clinical data. The experiments were designed knowledge. Therefore, the present case provided hint that the by QG and XZ. QG and XZ checked and approved the authen‑ hemolysis‑mediated mild hyperbilirubinemia may prepare and ticity of the raw data. All authors read and approved the final protect the liver from further injury. manuscript. In conclusion, the present study reported on a case of DILI, in the context of a mild form of rare congenital anemia resulting Ethics approval and consent to participate in bland cholestasis. Hemolysis‑induced mildly‑elevated unconjugated bilirubin may contribute to the milder DILI The present study was approved by the Ethics Committee observed in the current case compared to the previously of Ruijin Hospital (Shanghai, China; ID: 2016‑17). Written reported cases. Therefore, the present study highlighted the informed consent was provided by the patient. 6 HAN et al: CONGENITAL ANEMIA AND CHOLESTASIS

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